Present invention provides methods for initiating embryogenic cultures of plants. These methods provide somatic embryos needed for reproducing large numbers of plants by somatic embryogenesis. More specifically, the invention encompasses various methods and media compositions that can improve the frequency of initiation of embryogenic cultures.
The forest products industry is a major economic entity. In the United States the industry had sales of $400 billion dollars in 1994. Coniferous softwood species make up the majority of the trees harvested. In the Southeastern United States, Loblolly pine (Pinus taeda L.) and its close relatives are the most important species. In the Pacific Northwest, Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) is probably the most important commercial species. Likewise, in Europe, Norway spruce (Picea abies (L.) Karst.) is probably the most important conifer species.
Forest productivity can be increased by planting tree farms with large numbers of elite, high-quality trees. Unfortunately, trying to produce such trees by sexual reproduction yields seeds of unpredictable quality. Further, asexual reproduction by rooting vegetative cuttings, as is easily practiced with angiosperm species such as fruit trees, is not practicable for most coniferous species. What is needed is a method of clonally propagating large numbers of genetically superior conifer trees.
Plant tissue culture is the broad science of growing plant tissues on or in a nutrient medium containing minerals, sugars, vitamins and bioactive small molecules such as hormones. By adjusting the composition of the media, cultured tissues can be induced to grow or differentiate into specific cell types or organs. Somatic embryogenesis is a type of plant tissue culture where a piece of a donor plant is cultured such that an embryogenic culture is initiated. A proliferating embryogenic culture forms multiple embryos. An embryo is a discreet mass of cells with a well-defined structure that is capable of growing into a whole plant.
Somatic embryogenesis is widely used in a variety of species. In some species, somatic embryogenesis is used to propagate desirable plant genotypes. In many crop species, somatic embryogenesis is used to propagate tissues that have been genetically transformed, in order to regenerate whole transformed plants. Although somatic embryogenesis protocols are widely used and have been adapted to numerous species, most species include recalcitrant genotypes that are not readily regenerated. Further, some transformation methods, such as agrobacterium infection, electroporation and particle bombardment can damage plant cells and such damaged cells are not easily regenerated into whole plants. Thus, there is a need in the art for methods and compositions that can improve the efficiency of somatic embryogenesis in a wide variety of plant families.
Presently, somatic embryogenesis is seen as the most promising technology for the efficient multiplication of valuable coniferous germplasm. Since the late 1970""s, researchers have been working to develop methods of reproducing conifers by somatic embryogenesis. U.S. Pat. Nos. 4,957,866, 5,034,326, 5,036,007, 5,236,841, 5,413,930 5,491,090, and 5,506,136, herein incorporated by reference, describe various methods and media for conifer embryogenesis.
Culture initiation begins with the selection of a suitable explant, that is any plant cell, tissue or organ capable of forming an embryogenic culture. A typical explant in conifer somatic embryogenesis is the megagametophyte, also called the ovule or the female gametophyte, which is extracted from a pollinated female cone and which may contain single or multiple zygotic seed embryos. Next, an embryogenic culture is initiated from this explant by inducing cells within the explant to proliferate into a tissue mass containing at least one somatic early stage embryo. The successful establishment of such a culture is known as initiation.
Explants
Some conifer protocols known in the art use a megagametophyte that has been split open to expose the zygotic embryo or embryos. In one method, the embryo, while remaining attached to the megagametophyte by the long suspensor, is removed from the megagametophyte so that it is no longer surrounded by megagametophytic tissue. The embryo and attached megagametophyte are then both placed on a culture initiation media. In Douglas fir, for example, the embryo lies in a groove or channel within the megagametophyte. With careful manipulation, the embryo may be xe2x80x9cflipped outxe2x80x9d or displaced from the groove, yet ideally, remain attached to the megametophyte by the suspensor. Alternatively, the embryo or embryos may be completely separated from the megagametophye and placed on the media, either alone or next to the megagemetophytic tissue. The megagemetophytic tissue is often placed near the embryo, as it may produce growth regulators that promote initiation.
Extrusion
When intact conifer megagametophytes are used as the explant, the somatic embryogenesis process comprises a distinct step called extrusion. Prior to this distinct step in development of such megagametophytes, explants can be considered as pre-extrusion megagametophytes. Pre-extrusion therefore denotes the time before extrusion in such megagametophytes. Extrusion is the process in which a mass of embryogenic tissue is extruded from the micropylar end of the megagametophyte when it is placed on a suitable culture media. Becwar et al. (1990) observed in loblolly pine (Pinus taeda) that the extruded embryogenic tissue comes from cell division and proliferation in the suspensor region of the zygotic embryo or embryos. Extrusion can be observed using low power microscopy. The proliferation and growth of zygotic tissues pushes the embryogenic tissue out of the megagametophyte and it appears as a mass of filamentous suspensor-like cells containing single or multiple zygotic embryos. The mass sometimes contains dense globules that may be somatic proembryos or very early stage somatic embryos. Successful extrusion is scored whenever some tissue that has come outside of the intact megagametophytes via the micropyle is visible. Successful extrusion does not necessarily indicate that initiation will follow. Typically, many of the extruded masses of tissue fail to proliferate and initiate an embryogenic culture.
Many conifer somatic embryogenesis protocols known in the art use intact megagametophytes as a preferred explant. However, prior to the discovery of the present invention, the entire initiation process including both extrusion and initiation, were carried out in a single media.
Initiation
Successfully initiating a high percentage of embryogenic cultures requires the proper medium and culturing conditions. In conifers, an embryogenic culture is successfully initiated when the zygotic embryo or zygotic embryogenic tissue mass, which has been either extruded or physically removed from a megagametophyte, undergoes division and proliferation. A successfully initiated culture will consist of a whitish translucent mucilaginous tissue mass that contains pre-embryonal masses of cells, filamentous suspensor-like cells and early stage somatic embryos. In a successfully initiated culture, new somatic embryos can often be seen growing directly from the somatic embryos. Visualization of initiation is aided by the fact that zygotic embryos, as well as extruded tissues, often become brown while initiated tissues are whiter and more translucent. Initiated cultures contain from one to dozens of somatic embryos. Initiation is considered successful when at least one somatic embryo is visible. The appearance of at least two somatic embryos provides a useful confirmation of successful initiation.
After embryogenic cultures are initiated, a number of subsequent steps are required to regenerate plantlets. First, the initiated somatic embryos are transferred to a multiplication or maintenance medium with the right composition of plant hormones and other factors to induce the somatic embryos to multiply at a high rate. Cultures can multiply as fast as 2-6 times weekly. Once large numbers of embryos are obtained in the multiplication stage, the embryos are moved to a maturation and development medium. Here, the correct balance of plant hormones and other factors will induce the early-stage embryos to mature into late stage embryos. Following the maturation and development stage, embryos are germinated to form small seedlings. These seedlings are then acclimated for survival outside of the culture vessel. After acclimation, the seedlings are ready for planting.
As noted above, protocols known in the art utilize a single media to induce the extrusion of embryogenic tissue and to initiate the proliferation of this tissue. The composition of the culture medium will determine the efficiency of plant regeneration by somatic embryogenesis. Several media constituents are known in the art to influence somatic embryogenesis in conifers.
Gelling Agent
The effect of gelling agents in plant tissue culture media has been investigated in various systems. Gelling agents are compounds that convert liquid media into solid or semi-solid colloidal suspensions. Increased amounts of gelling agent make the media more solid and decrease media matric potential, which is a measure of the ease with which water is extracted from the gel. One report suggests that the effect of matric potential on total media water status is negligible (Taito et al., 1999). Conversely, other reports suggest that changes in matric potential due to increased gelling agent have a significant effect on water availability, and thus affect the growth of cultured plant cells. For example, Ghashghaie et al. (1991) studied the effect of gelling agent concentration on shoot cultures of Rosa hybrida and found that water availability and shoot elongation increased with decreasing agar concentration. Similarly, Owens and Wozniak (1991) reported that increasing concentrations of gelling agent decreased gel matric potential and negatively affected somatic embryo and shoot growth. Etienee et al. (1991) observed that embryogenic culture initiation in rubber tree (Hevea brasiliensis) was increased using liquid media as opposed to gelled media.
Few studies have investigated the effects of the amount or composition of gelling agents in conifer embryogenic initiation media. VonArnold (1987) observed no effect of gel stiffness on initiation in Picea abies. Tremblay and Tremblay (1991), in two Picea species, compared gelling agents and obtained better initiation using GELRITE than comparable amounts of agar. Harry and Thorpe (1991) also found GELRITE to be superior to other gelling agents in initiating Red Spruce (Picea rubens) embryogenic cultures. None of these studies or other methods known in the art utilized initiation media with less than 1 g/l GELRITE or 4 g/l agar. For example, in U.S. Pat. No. 5,236,841, Gupta and Pullman disclose solid initiation media containing 6.0 and 7.0 g/l agar. Similarly, U.S. Pat. No. 5,413,930 and 5,506,136, herein incorporated by reference, disclose media using no less than 1 g/l GELRITE and 4 g/l agar.
As suggested above, all conifer embryogenic culture initiation methods known in the art utilize a solid initiation media. In contrast, liquid media are commonly employed in later culturing and regeneration steps. For example, in U.S. Patent 5,236,841, Gupta and Pullman disclose a method of regenerating coniferous plants by somatic embryogenesis that uses liquid maintenance and development media. Similarly, in U.S. Pat. No. 5,491,090, herein incorporated by reference, Handley et al. disclose a post-induction liquid media used to maintain embryogenic cultures.
While liquid media is used in later stages of conifer somatic embryogenesis, the use of liquid media in initiation media has not been suggested. Rather, conifer embryogenic initiation media known in the art contain sufficient gelling agents such as GELRITE, agar, gellan gum and agarose to produce a solid or semi-solid media.
The development of a liquid initiation media would be particularly advantageous as liquid media are more amenable to automation (Majada et al., 1991). Large scale germplasm screening and plant propagation will depend upon economical methods, and automation can reduce labor costs and increase the efficiency of somatic embryogenesis (Gupta et al., 1999).
Activated Charcoal
Activated charcoal or other adsorbent materials have been widely used in plant tissue culture media where they are believed to function as an adsorbent for toxic metabolic products and undesirable amounts of residual hormones. In one early report, Johansson (1983) measured embryogenesis in cultured anthers of ornamental plant species and utilized liquid media overlaying solid media that contained activated charcoal. He concluded that activated charcoal was effective in removing inhibitory amounts of ABA and other undesirable materials such as phenolics from the liquid media.
In conifer somatic embryogenesis, activated charcoal has been used in the development and maturation media. For example, U.S. Pat. No. 5,036,007, herein incorporated by reference, discloses a method of regenerating conifers using 2000 mg/l activated charcoal in the development media where early stage somatic embryos are matured. The use of activated charcoal in the initiation medium is also known. For example, U.S. Pat. Nos. 5,294,549, herein incorporated by reference, and U.S. Pat. No. 5,236,841, disclose initiation media containing 2500 mg/l activated charcoal.
Abscisic Acid
The use of abscisic acid (ABA) in culture media is known in the art of conifer tissue culture. Typically, ABA is used in the later stages of somatic embryogenesis following culture initiation. For example, Salajova et al. (1999), working with Pinus nigra, reports the use of ABA in the development media. Recently, it has been shown that ABA is also effective in aiding initiation of Pinus embryogenic cultures. U.S. Pat. No. 5,677,185, and U.S. Pat. No. 5,856,191, both herein incorporated by reference, disclose solid initiation media containing ABA and report initiation frequencies of 30-38% using media containing 30-90 mg/l ABA.
Silver Nitrate
Ethylene (C2H4) is a gaseous plant growth regulator that often accumulates during plant tissue culture. Ethylene is generally considered to depress the growth of cultured tissues. In conifers, however, some workers have observed that ethylene reduces the growth of cultured Picea embryogenic tissues (Kumar et al., 1989) while others have found no effect (Kvaalen, 1994). Several compounds, such as silver nitrate (AgNO3) and cobalt chloride (CoCl2), have been identified that block the action of ethylene.
The use of silver nitrate or other ethylene inhibitors is known in the art of conifer somatic embryogenesis but reports of its efficacy are mixed. Abdelmalek at al. (1999) tested the effects of silver nitrate on maturation of Black spruce (Picea mariana) somatic embryos and concluded that silver nitrate did not affect maturation and growth of the cultures, whereas Kong and Yeung (1995) obtained the opposite result in White spruce (Picea glauca). In Blue spruce (Picea pungens), Afele and Preveen (1995) observed that silver nitrate inhibited induction of embryogenic cultures. In contrast, Li and Huang (1996) observed that silver nitrate, at concentrations of about 29-59 xcexcM improved embryogenic culture initiation in loblolly pine.
In some varieties of conifer, methods exist for the efficient initiation of embryonic cultures. For example, a review by Tautorus et al. (1991) shows that numerous workers in the field have obtained Picea initiation frequencies over 50% and as high as 95%. In Norway Spruce (Picea abies), Chalupa (1997) obtained initiation frequencies of 52-96%.
Despite the success in propagating spruce (Picea) species and Douglas fir by somatic embryogenesis, the propagation of Pinus species is much more difficult. Many Pinus species, including Loblolly pine (Pinus taeda) do not readily initiate embryonic cultures. Typical initiation frequencies of about 1-12% are reported for various Pinus species (Becwar et al. 1988, Jain et al. 1989, Becwar et al. 1990 Li and Huang 1996). Laine and David (1990) however, were able to obtain high frequencies of initiation (up to 59%) in Pinus caribaea, suggesting that not all Pinus species are recalcitrant. Also, one earlier report described initiation frequencies of 54% in White pine (Pinus strobus) (Finer et. al, 1989). However, other workers were not able to duplicate this success (Michler et al., 1991). The results in the literature demonstrate the recalcitrance of Pinus species, especially Loblolly pine, in regeneration by somatic embryogenesis. One problem is that there is great variability among different genotypes in amenability to tissue culture. Consequently, regeneration potential can vary dramatically from tree to tree, and in open pollinated cones, from seed to seed even on a single tree. This complicates efforts to screen germplasm, because current methods used to regenerate plants for evaluation will select genotypes best able to survive the culturing process rather than a broad range of genotyopes.
Recently, some progress has been made in increasing initiation frequencies in recalcitrant Pinus species. Recent patents disclose improved methods of initiating embryonic cultures in Lobolly pine. U.S. Pat. No. 5,413,930, hereby incorporated by reference, describes the use of gelled initiation media to attain average initiation frequencies of 17%. U.S. Pat. No. 5,506,136, hereby incorporated by reference also discloses a method incorporating a gelled media and reports initiation frequencies of 15% and 50% in two different genotypes. U.S. Pat. No. 5,677,185 and U.S. Pat. No. 5,856,191, both hereby incorporated by reference, disclose solid initiation media containing abscisic acid (ABA) and report initiation frequencies of 30-38%.
Thus, there is a need in the art for further progress in the efficiency of culture initiation. There is a need in the art for a somatic embryogenesis method that yields high rates of initiation for multiple and diverse genotypes. Such high rates are important when selecting superior trees, the growth characteristics of many different genotypes, as trees growing in the field, must be evaluated. If one is attempting to propagate several genotypes and initiation frequencies are low, the majority of the material cannot be evaluated. Even assuming that desirable growth characteristics are not linked to embryonic culture potential, at initiation rates below 10%, over 90% of the desirable genotypes will be lost. Further, methods that select only the most easily propagated fraction of the population may actually select against other desirable traits.
There is a need in the art for an efficient somatic embryogenesis regeneration system for conifers and other plants to be used in conjunction with plant transformation techniques. The genes that control tree quality are currently being identified and characterized. In order to develop transgenic trees incorporating these genes, the existence of a reliable tissue culture and regeneration system is vital. Further, there is a need in the art for a regeneration system that can be automated for the large scale production of high-quality or transformed seedlings.
It is an object of this invention to provide novel methods to improve the efficiency of somatic embryogenesis in plants and, in particular, to provide efficient methods of initiating embryogenic cultures of diverse coniferous species and diverse genotypes within a species, specifically including diverse Pinus genotypes such as Loblolly pine (Pinus taeda). It is a further object to provide a method of somatic embryogenesis that will dependably and consistently provide coniferous plantlets in large quantities. It is yet another object to provide a general method of somatic embryogenesis that can dependably and consistently reproduce large numbers of clones of selected individuals of forest species that heretofore have been recalcitrant using known methods for somatic embryogenesis. It is still a further object to provide a method whereby superior genotypes of coniferous trees can be multiplied by tissue culture in the large quantities needed for reforestation. It is still a further object to provide a liquid media method that facilitates automation and large scale reproduction. It still another object to provide a method that generates robust somatic embryos capable of withstanding extended periods of cold storage or cryogenic preservation.
These and many other objects will become readily apparent to those skilled in the art by reading the following detailed description.
The present invention is specifically directed to the use of various compositions and methods to improve the frequency of embryogenic culture initiation. The compositions and methods are particularly advantageous for the production of thousands of somatic embryos from multiple and diverse genotypes. The production of high numbers of somatic embryos aids in the efficient conversion of embryos into plants growing in soil. Thus, these methods allow the inclusion of more genotypes in subsequent clonal field tests and thereby increase the likelihood of being able to select highly productive genotypes. Further, some of the methods disclosed provide the art with a liquid initiation media, which allows the preliminary steps in somatic embryogenesis to be more easily automated and adapted to large scale production. In addition, use of these methods generates early stage embryos that can be retained for extended periods of time in cryogenic storage. Further, the methods interface very well with genetic engineering techniques for mass production of clones of genetically modified genotypes.
Several embodiments of the present invention are directed to the propagation of conifers. However, embodiments of the invention can be broadly applied to all plant families. As set forth below, these broadly applicable embodiments can increase the efficiency of embryogenic culture initiation in a wide variety of species and one of ordinary skill in the art will readily recognize the advantages of applying these compositions and methods to diverse plant families.
Many embodiments of the present invention are generally suitable for reproducing woody gymnosperms of the order Coniferales. These are particularly well suited for generating clones of superior forest trees for reforestation, including species within the families Pinaceae, Cupressaceae, and Taxodiaceae. Most particularly, all species within the genera Abies, Pinus, Picea, Tsuga, Pseudotsuga, Thuja, Juniperis, Larix, Taxus and Sequoia are amenable to multiplication using the disclosed methods and compositions.
Some embodiments of the present invention are especially applicable to somatic tissue obtained from the Pinus species including, but not limited to, the following: Pinus taeda (loblolly pine), Pinus elliottii (slash pine), Pinus palustris (longleaf pine), Pinus serotina (pond pine), Pinus radiata (Monterey pine), and Pinus rigida (pitch pine). In addition, embodiments of the current invention are specifically applicable to hybrids (i.e., interspecies hybrids) of the above mentioned pines, including crosses between Pinus rigida and Pinus taeda, crosses between Pinus serotina and Pinus taeda, and reciprocal crosses.
Specific aspects of the invention are applicable to a broad number of plant families, including but not limited to the grass family (Poaceae), legumes (Fabaceae), plants from the mustard family (Brassicaceae), plants from the nightshade family (Solanaceae), the rose family (Rosaceae) and other plant families containing food, forest, forage, fiber or industrial crop species. For example, embryogenic culture initiation can be improved in important crops from the grass family, such as rice (Oryza sativa), corn (Zea mays) and wheat (Triticum spp.) using specific embodiments of the invention. Likewise, these embodiments can be applied to improve somatic embryogenesis in important legume crops such as soybean (Glycine max) or lentil (Lens culinaris).
Any plant tissue explant capable of being employed for somatic embryogenesis is suitable for use in the present invention. A number of explant sources have been used successfully in somatic embryogenesis . These include, but are not limited to, tissue from cotyledons, hypocotyls, epicotyls, buds, meristematic centers from buds or roots, tissues extruded from megagametophytes, and seed embryos. In conifers, one can use an immature whole megagametophyte containing zygotic embryos or an isolated immature dominant zygotic embryo as the explant. Zygotic embryos removed from seeds can be used. These may or may not include the surrounding gametophyte.
One embodiment of the invention comprises the use of separate media for extrusion and initiation. One aspect of this method comprises the use of a solid media upon which ovules are cultured until an optimal level of extrusion has occurred. At this point, a liquid initiation media is poured over the solid media and the extruded tissues are cultured until they have initiated embryogenic cultures. Alternatively, the extruded tissue may be removed from the extrusion media and placed in liquid initiation media.
The processes of the present invention is not limited to any single culture medium. Any of a number of well known media, such as that of Murashige and Skoog (1962), may be used. However, the present inventors have found the basal medium described in Table 1 to give excellent results, particularly when used for culturing loblolly pine (Pinus taeda).
The extrusion or initiation medium will normally be one of those well known from past work which contains a balanced concentration of inorganic salts and organic nutrient materials. The media may contain plant hormones including auxins and cytokinins. What constitutes an effective amount of auxin or cytokinin will depend upon the species and the type of media used. For example, U.S. Pat. No. 5,565,355, herein incorporated by reference, discloses that Pinus radiata embryogenic cultures may be initiated in media that contains no auxin, cytokinin or other similar plant growth regulators. Therefore, an effective amount of auxin or cytokinin to be used in the invention can be zero. However, one can also incorporate auxin and cytokinin into the media of the present invention. Suitable levels for the present method include about 0.1 to 120 mg/l for auxin and about 0.1 to 100 mg/l for cytokinin.
The particular auxins and cytokinins used and their exact concentrations, or whether they are used at all, will depend somewhat on the species being cultured and even on the particular genotype within that species. For example, in Douglas fir, a suitable concentration of auxin is 110 mg/l and a suitable concentration of cytokinin is 88 mg/l. In solid media used for Loblolly pine, a suitable concentration of auxin is 2.0 mg/l and a suitable concentration of cytokinin is 1.24 mg/l. In liquid media used for Loblolly pine, a suitable concentration of auxin is 0.3 mg/l and a suitable concentration of cytokinin is 1.24 mg/l. The optimal amount to be used can be readily determined empirically by one of ordinary skill in light of the present disclosure. Of course, the optimal level of growth hormones also depends on the presence or absence of an adsorbent material, such as activated charcoal. Activated charcoal can adsorb a large proportion of free auxins and cytokinins, requiring the addition of higher levels to compensate. Finally, it will be understood by those skilled in the art that analogs and similar plant hormones may be substituted for the auxins and cytokinins listed below in Table 2.
The media should contain a readily metabolized carbohydrate energy source, like a sugar such as maltose, glucose, fructose, sucrose, or galactose, or combinations thereof. One may utilize a wide range of sugar concentrations, such as between 5 and 70 g/l. Maltose is an effective carbohydrate source. Several embodiments of the present invention utilize media containing maltose at a concentration of 15 g/l.
If a solid, semi-solid or viscous initiation media is desired, gelling agents can be added to the initiation medium. For example, the use of 2.5 to 8 g/l of agar, 0.5 to 5.0 g/l of gellan gum, 3.0 to 8.0 g/l of agarose will yield a semi-solid to solid media, with gel stiffness increasing as the level of gelling agent is increased. Several embodiments of the present invention utilize solid media containing 2.0 g/l gellan gum.
An improvement of the present invention is the use of an initiation media that contains little or no gelling agent. Indeed, heretofore no one has shown that using completely liquid media in conifer embryogenic culture induction is effective in initiating conifer embryogenic cultures. Some embodiments of the present invention comprise the use of a liquid initiation media containing from about 0.0-2.5 g/l agar, 0.0-0.5 g/l gellan gum, 0.0-3.0 g/l agarose or 0.0-1.5 g AGARGEL, including any concentration of agar, gellan gum, agarose or AGARGEL subscribed by the recited ranges. Irrespective of its composition, a liquid initiation media is considered liquid, within the scope of the invention, if an explant does not remain on the surface of the media throughout normal culture conditions. Alternatively, a liquid media is hereby defined as one which can be poured into a culture vessel and form a substantially level surface (disregarding the meniscoid effects of surface tension) at 20xc2x0 C. One embodiment of the liquid initiation media contains no gelling agent at all.
It will be understood by those skilled in the art that the gelling agents listed above are representative and that the use of equivalent substitutes falls within the scope of the invention. Further, the invention encompasses the combination of different gelling agents such that the desired solid, semi-solid or liquid media is formed.
This invention improves on the prior art by teaching the use of reduced levels of activated charcoal in the extrusion and the initiation media. Conifer somatic embryogenesis methods known in the art utilize much larger amounts of activated charcoal. For example, U.S. Pat. Nos. 5,294,549 and 5,236,841 disclose initiation media containing 2500 mg/l activated charcoal. High initiation frequencies can be attained using less than 2500 mg/l, for example, between about 10 to 2000, 10 to 650, or 100 to 500 mg/l of activated charcoal, or any concentration subsumed within those ranges. Activated charcoal may be used in either liquid or solid media. An effective activated charcoal concentration for Loblolly pine initiation is about 10 to about 100 mg/l. A concentration of 25 to 75 mg/l may be used. Several embodiments of the invention employ a concentration of 50 mg/l. Several embodiments of the initiation media used for Norway spruce contain 200 to 400 mg/l activated carbon. A concentration of 250 to 350 mg/l may be used. One embodiment of the invention utilizes a concentration of 300 mg/l. Those skilled in the art will recognize that other adsorbent materials may be substituted for activated charcoal, including other forms of activated carbon.
Activated charcoal adsorbs large amounts of copper and zinc in the media, and this depletion may result in deficiencies. The depletion of zinc and copper caused by activated charcoal can be compensated by the addition of extra zinc and copper. Working with multiple conifer species, we have discovered that when copper and zinc are added to the media to compensate for the adsorbed ions, the growth of embryonic cultures is enhanced. A suitable level of anhydrous ZnSO4 is 14.7 mg/l in a liquid media containing 50 mg/l activated charcoal. A suitable level of anhydrous CuSO4 is 0.17 mg/l in a liquid media containing 50 mg/l activated charcoal. Those skilled in the art will recognize that several alternative sources of zinc and copper may be substituted for those suggested above.
The present invention comprises novel methods of using abscisic acid (5-(1-hydroxy-2,6,6,-trimethyl-4-oxo-2-cyclohexen-1-yl)-3-methyl-2,4-penta dienoic acid) (ABA) to improve induction of embryogenic cultures. In one embodiment, explants are pre-treated for several minutes up to overnight with an aqueous solution of abscisic acid prior to culturing with an initiation media that lacks ABA. The duration of the pre-culturing soak and the concentration of ABA to be used in the soaking solution may vary with the type of explant used and the permeability of the explant to the solution, and explants may be rinsed with media lacking ABA prior to culturing. Several methods of the current invention utilize ovules excised from conifer seeds. In one embodiment, the ovules are soaked in water, buffer or liquid media containing from 1-50 parts per million (ppm) ABA. Some embodiments of the invention utilize a concentration of 10-20 ppm ABA in the soaking solution. Some embodiments of the pre-treatment method utilize a soaking duration of one to 80 minutes. Alternatively, one can pre-treat for 15 to 120 minutes or 30 to 45 minutes.
Several embodiments of the invention employ a liquid media containing ABA. When liquid initiation media is used, the media can contain from 0.01 to 1 mg/l ABA. In one embodiment of the liquid media, the media contains from 0.05 to 0.15 mg/l ABA. Some embodiments of the liquid media contain 0.1 mg/l ABA. In contrast, in some embodiments of the invention, the media is substantially free of ABA, meaning that ABA is effectively absent from the media formulation. In such embodiments, ABA is not present at all, or is present in insignificant amounts, such that plant growth in such media is indistinguishable from growth in the complete absence of ABA.
Liquid initiation media containing ABA can be used in numerous ways. In one embodiment of the invention, all culturing takes place in a liquid medium containing ABA. In a separate embodiment, the invention comprises the use of liquid initiation media containing ABA to initiate embryogenic cultures from tissue that has been previously extruded on a separate extrusion media containing little or no ABA. In various embodiments of this method, the extrusion media contains less than 0.1 mg/l ABA, less than 0.01 mg/l ABA, or is completely lacking ABA (0.00 mg/l). Extrusion occurs on this media. After extrusion has occurred, a liquid media containing ABA is poured over the extruded tissue or the tissue is removed from the solid media and placed in the liquid media. It will be recognized by those skilled in the art that ABA can have physiological effects at very low concentrations. It will also be recognized by those skilled in the art that the method of ABA application, such as vacuum filtration, soaking or physical contact with culture media, will affect the ABA concentration experienced by the explant. Further, the ABA delivery media, whether it is solid media, liquid media or buffer, will also affect the concentration experienced by the explant. Additionally, the presence of an adsorbent material such as activated carbon can greatly reduce the amount of free ABA such that additional ABA must be added to the medium to compensate. Therefore, it is understood that what constitutes an effective amount of ABA will depend on the method of application and media composition. Using the methods disclosed in the Examples, one of skill in the art can readily determine empirically what will constitute an effective amount for each protocol. Further, it will be understood by those of skill in the art that the invention encompasses the use of analogs and variants of ABA.
One aspect of the present invention is the use of sliver nitrate (AgNO3) in the extrusion and initiation media. It is known in the art that silver nitrate decreases the action of ethylene and can have significant effects in plant tissue cultures. The use of silver nitrate in conifer embryogenic culture initiation media is known. However, there are conflicting results in the art as to whether it improves or inhibits somatic embryogenesis in conifers. Nevertheless, our results indicate that silver nitrate improves embryogenic culture initiation. Those skilled in the art will recognize that other ethylene inhibitors such as silver thiosulfate ([Ag(S2O3)2]3xe2x88x92) or cobalt chloride (CoCl2) may be substituted for silver nitrate. Silver nitrate may be used in liquid or solid media, at concentrations ranging from 1-50 xcexcM. When silver nitrate is used in a liquid media concentrations of 10-20 xcexcM, or about 2-4 mg/l are effective.
This invention improves on the plant tissue culture art by teaching the novel use of brassinosteroids in somatic embryogenesis. We have discovered that brassinosteroids can be used to promote initiation of embryogenic cultures. The brassinosteroids are a group of naturally occurring steroidal lactones which include brassinolide and its analogs. The brassinosteroids were relatively recently discovered to be plant growth regulators. In angiosperm species, brassinosteroids have been shown to have diverse, tissue-specific and species-specific effects, including the stimulation of cell elongation, stimulation of ethylene production and increasing resistance to abiotic stress (Brosa, 1999). Brassinolide is a brassinosteroid found in many plant species. The formula of brassinolide is: 
Brassinolide analogs are known in the art. For example, U.S. Pat. No. 5,965,488, herein incorporated by reference, discloses the following general formula for brassinolide analogs: 
wherein R4 and R5 are C1-C6 lower alkyl groups. The C1-C6 alkyl groups represented by R4 and R5 are preferably C1-C4 straight-chain alkyl groups and include a methyl group, ethyl group, propyl group and butyl group. In particular, the ethyl group and propyl group are preferable for high activity.
Naturally occurring brassinosteroids have been isolated from conifers (Kim et al., 1990). Brassinosteroids have been applied exogenously to pine seedlings (Sasse et al., 1992) and spruce cuttings (Ronsch et al., 1993) and have been found to promote root growth. Nevertheless, the use of brassinosteroids in conifer tissue culture has not been previously reported. In fact, the effects of brassinosteroids on cultured plant cells of any species has not been extensively investigated. In one of the few reports available on the effects of brassinosteroids in tissue culture, Yang et al. (1999) found slightly increased growth of cultured cells of Onosma paniculatum (an angiosperm species used as an herbal medicine) in media containing 0.01 ppb brassinolide. Conversely, Roth et al. (1989) observed that brassinosteroids inhibited the growth of cultured tobacco cells. Both of these reports dealt with non-embryogenic cell cultures. The use of brassinosteroids in somatic embryogenesis has not been reported.
As noted above, heretofore no one has explored the efficacy of brassinosteroids at any stage of the somatic embryogenesis process, for any species. Consequently, heretofore no one even suggested that using brassinosteroids is highly advantageous in initiating embryogenic tissue cultures. This invention thus comprises the use of brassinosteroids in somatic embryogenesis, in any species, including conifers. The use of brassisteroids could potentially be employed in somatic embryogenesis of Conifer species within the genera Abies, Pinus, Picea, Tsuga, Pseudotsuga, Thuja, Juniperis, Larix, Taxus and Sequoia. Additionally, brassinosteroids can be used in somatic embryogenesis of species in important families such as Poaceae, Fabaceae, Brassicaceae, Solanaceae, Rosaceae, and other plant families containing food, forest, forage, fiber or industrial crop species.
The present invention comprises the use of brassinosteroids in liquid or solid initiation media. Additionally, in conifers, brassinosteroids can be used in extrusion media. The results of our experiments showed that brassinolide has a positive effect on the initial proliferation of embryogenic tissues in plants. In our media, brassinolide was effectively used in concentrations ranging from 0.005-0.25 xcexcM. However, it will be recognized by those skilled in the art that the presence of adsorbent materials such as activated carbon can potentially reduce the amount of free brassinolide available to cultured tissues. Thus, the concentration that represents an effective amount of brassinolide will depend upon the media composition, as well as the form of the media (liquid vs. solid) as well as the method of application. Using the Examples disclosed in this application, one of skill in the art can readily determine what constitutes an effective amount of brassinosteroid in a specific media. One embodiment of the invention comprises a liquid media containing about 0.10 xcexcM brassinolide. It will be recognized by those skilled in the art that other brassinosteroids, including brassinolide analogs, may be substituted for brassinolide, for example those disclosed in U.S. Pat. No. 5,965,488, which is herein incorporated by reference.
Numerous studies have focused attention on the composition of the atmosphere within plant tissue culture vessels. Consequently, it is known in the art that the composition of the atmosphere within culture vessels can have significant effects on somatic embryogenesis. Cultured cells deplete oxygen, and produce carbon dioxide and ethylene, which can accumulate in the headspace above the media in covered culture vessels. The effects of these atmospheric changes have been studied in several species, including conifers. For example, in Picea abies, Kvaalen and von Arnold (1991) observed that a low oxygen atmosphere stimulated the initiation of embryogenic cultures when full strength media was used, and inhibited initiation when half strength media was used. Selby et al. (1996) studying Sitka spruce (Picea sitchensis), determined that removing volatile compounds from the atmosphere of the culture vessel stimulated embryo maturation. Abdelmalek et al. (1999) observed that Picea mariana embryogenic cultures had better maturation rates in unvented containers than vented containers, and ruled out ethylene accumulation as causing this effect.
Despite the attention given to the composition of environmental gasses, the effect of increased atmospheric pressure on cultured cells has not been widely investigated in plants. This is unlike the situation in animal tissue culture, where the effects of hydrostatic pressure on various cell types has been extensively studied; usually the pressure is applied to simulate mechanical stress in the cells"" normal environment and to measure effects on factors such as gene expression or cell differentiation. In the field of plant tissue culture, only a single reference describes the effects of culturing cells in a pressurized environment. Specifically, Barthou et al. (1997) studied sunflower (Helianthus annuus L.) protoplasts cultured for 7 days at 2-6 times normal atmospheric pressure. Barthou reported a generally adverse effect on cell growth, observing that pressures above 4 atmospheres inhibited cell division and microtubule synthesis. Thus, the sole reference in the field teaches away from the use of increased pressure in plant tissue culture.
The present invention improves the art of plant tissue culture by teaching the use of elevated atmospheric pressure in somatic embryogenesis. As noted above, heretofore no one has explored the efficacy of using increased atmospheric pressure at any stage of somatic embryogenesis. Indeed, heretofore no one has shown or even suggested that using increased atmospheric pressure is highly advantageous in initiating embryogenic tissue cultures. The results of our experiments showed that increased atmospheric pressure within the culture vessel has a profound effect on the initial proliferation of embryogenic tissues in plants. Our experiments also showed that increased atmospheric pressure increases the extrusion of embryogenic tissues in conifers. The improvement of the present invention comprises culturing embryogenic tissue, using either liquid, semi-solid or solid initiation media, and increasing the atmospheric pressure of the culture environment to above normal pressure ( greater than 1 atm) for all or part of the initiation period. Our results demonstrate that the pressure effect does not depend on continuous pressure. Occasional depressurization so that culture dishes can be observed does not appear to diminish the beneficial pressure effect. In one embodiment, increased atmospheric pressure is applied during the entire extrusion and initiation process. In other embodiments, increased pressure is applied for at least one week, or from 2 to 14 weeks, or from 3 to 10 weeks. In one embodiment the pressure ranges from 1.1 to 5.0 atmospheres. In another embodiment the pressure is 1.3 to 1.7 atmospheres. In yet another embodiment, the pressure is 1.5 atmospheres. As used herein, 1 atmosphere is generally equivalent to about 1013 millibar, however, the increased pressure range of the invention may also be calculated as pressure in excess of ambient atmospheric pressure.
Culturing during the extrusion or initiation stage may be carried out in the dark, under very low light conditions, or in full light until an embryogenic mass forms. In general, initiation in full dark is preferred. Culture initiation lasts for a period of from about 2 to 14 weeks. In another embodiment the culture period is from 3-10 weeks.
The media within a vessel may be replaced. Occasionally, a culture that has initiated and contains somatic embryos will not proliferate vigorously. If such a culture is in liquid media, the addition of fresh liquid media may promote growth and lead to more vigorous initiation. Alternatively, if such a culture is on solid media, the tissue can be moved to new solid media in a different vessel or can be moved to a new location in the same vessel. One of skill in the art will recognize that the methods of the invention can be carried out wherein old media is periodically replaced with fresh media of the same composition.
A number of terms are known to have differing meanings when used in the literature. The following definitions are believed to be the ones most generally used in the field of botany and are consistent with the usage of the terms in the present specification.
xe2x80x9cAuxinsxe2x80x9d are plant growth hormones that promote cell division and growth.
xe2x80x9cCorrosion cavityxe2x80x9d is the cavity within the megagametophyte tissue of conifers formed by the growth and enlargement of the zygotic embryos.
A xe2x80x9ccotyledonary embryo,xe2x80x9d sometimes simply referred to as an xe2x80x9cembryo,xe2x80x9d has a well defined elongated bipolar structure with latent meristematic centers having clearly visible cotyledonary primordia surrounding and usually obscuring an apical dome at one end and a latent radicle at the opposite end. The cotyledonary structure frequently appears as a small xe2x80x9ccrownxe2x80x9d at one end of the embryo. A cotyledonary somatic embryo is analogous to a zygotic embryo.
xe2x80x9cCryopreservationxe2x80x9d refers to the common process of storing cultures at ultra-low temperatures for future use.
xe2x80x9cCytokininsxe2x80x9d are plant growth hormones that affect the organization of dividing cells.
An xe2x80x9cEmbryo,xe2x80x9d depending on its stage of development, will have a variable morphology. Stages 1-9.10 as defined by Pullman and Webb, TAPPI RandD Division 1994 Biological Sciences Symposium, pp 31-34, which is hereby incorporated by reference, define embryos at different points of development. Development spans from Stage 1, where embryos are composed of 12 or less cells to Stage 9.10 where embryos are well developed and have accumulated their full mature weight.
xe2x80x9cEmbryogenic tissuexe2x80x9d in Conifers is a translucent white mucilaginous mass that contains early stage embryos and suspensor-like cells, and may contain small, dense globular clusters of cells capable of forming somatic embryos.
An xe2x80x9cexplantxe2x80x9d is the organ, tissue, or cells derived from a plant and cultured in vitro for the purpose of starting a plant cell or tissue culture.
xe2x80x9cExtrusionxe2x80x9d is the process by which zygotic embryos and/or embryogenic tissue derived from zygotic embryos emerges or extrudes from the corrosion cavity of the megagametophyte of conifer seeds via the opening in the micropylar end, when placed in culture.
xe2x80x9cGenotypexe2x80x9d is the particular genetic composition of an organism.
xe2x80x9cInitiationxe2x80x9d is the initial cellular proliferation and development of zygotic tissues to form a culture containing somatic embryos.
A xe2x80x9cmegagametophytexe2x80x9d is haploid nutritive tissue of the conifer seed, of maternal origin, within which the conifer zygotic embryos develop.
A xe2x80x9cmicropylexe2x80x9d is the small opening in the end of the conifer seed from which zygotic and/or embryogenic tissue extrudes from the megagametophyte when cultured.
xe2x80x9cNutrientsxe2x80x9d are the inorganic nitrogen, inorganic minerals, vitamins, organic supplements, and carbon sources necessary for the nourishment of the culture.
A xe2x80x9cplantletxe2x80x9d is a small germinating plant asexually reproduced by tissue culture.
A xe2x80x9csomatic embryoxe2x80x9d is a vegetatively produced embryo formed during culturing.
xe2x80x9cSomatic embryogenesisxe2x80x9d is the process using tissue culture techniques for generating multiple embryos from an explant. The embryos generated from a given tissue source are believed to be genetically identical.
A xe2x80x9csuspensor cellxe2x80x9d is an elongated and highly vacuolated filamentous cell.
A xe2x80x9czygotic embryoxe2x80x9d is an embryo(s) which is derived from the sexual fusion of gametes during pollination and is found within the megagametophyte.
The present invention is illustrated by the following Examples, which are not intended to be limited in any way.